JP5707132B2 - Copy number variation determination, method and system - Google Patents

Abstract

The present invention methods and systems for determining copy number variation of a target polynucleotide in a genome of a subject including amplification based techniques. Methods can include pre-amplification of the sample followed by distribution of sample and a plurality of reaction volumes, quantitative detection of a target polynucleotide and a reference polynucleotide, and analysis so as to determine the relative copy number of the target polynucleotide sequence in the genome of the subject.

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application is a US Provisional Patent Application No. 60 / 967,897 filed Sep. 7, 2007 under US Patent Act §119 (e). The benefit of which is incorporated herein by reference.

The present invention relates to methods for determining copy number variation in the genome from small populations or individuals and is useful for biological and medical applications.

“Digital PCR” refers to a method in which individual nucleic acid molecules present in a sample are distributed into a number of separate reaction volumes (eg, chambers or aliquots) prior to PCR amplification of one or more target sequences. . Individual molecules in a sample so that after reaction each reaction volume contains less than one separate polynucleotide molecule (or aggregate of linked polynucleotide molecules) and most chambers contain zero or one molecule. The concentration of is adjusted. The amplification of the target sequence results in a binary digital output where each chamber is identified as containing or not containing a PCR product indicating the presence of the corresponding target sequence. The number of reaction volumes containing detectable levels of PCR end product is a direct measure of absolute nucleic acid content. In one digital PCR version, polynucleotide molecules are distributed by dividing them into separate reaction volumes. One splitting method uses BioMark ™ 12.765 Digital Array (Fluidigm Corp., South San Francisco, Calif.). The chip utilizes integrated channels and valves that divide the sample and reagent mixture into 765 nanoliter volume reaction chambers. The DNA molecules in each mixture are randomly divided into 765 chambers in each panel. The chip is then temperature cycled and imaged with Fluidigm's BioMark real-time PCR system, and positive chambers originally containing one or more molecules can be counted by digital array analysis software. For a discussion on digital PCR, see, for example, Non-Patent Document 1; McBride et al., Patent Document 1, especially Example 5.

  Copy number variation (CNV) is an amplification or deletion of a genomic region with a size of 500 bases or more (often between 5,000 and 5 million bases). Genome-wide studies have revealed the existence of numerous CNV regions in humans and extensive genetic diversity in the general population. CNV has been the focus of much research in recent years due to its role in human genetic disorders. For example, see Non-Patent Document 2, Non-Patent Document 3, Non-Patent Document 4, Non-Patent Document 5, Non-Patent Document 6, and Non-Patent Document 7, each of which is incorporated herein by reference. Aneuploidy, such as trisomy or total chromosomal deletion, is a limited type of copy number variation associated with various human diseases.

US Patent Application Publication No. 2005/0252773

Vogelstein and Kinzler, Proc Natl Acad Sci USA (1999) 96: 9236-41 Iafrate et al., Detection of large-scale variation in the human genome. Nat Genet (2004) 36: 949-951. Sebat et al., Large-scale copy number polymorphism in the human genome. Science (2004) 305: 525-528. Redon et al., Global variation in copy number in the human genome. Nature (2006) 444: 444-454. Wong et al., A complete analysis of common copy-number variations in the human genome. Am J Hum Genet (2007) 80: 91-104 Ropers, New perspectives for the elucidation of genetic disorders. Am J Hum Genet (2007) 81: 199-207 Lupski, Genomic rearrangements and spatial disease. Nat Genet (2007) 39: S43-S47

  The present invention relates to a method for determining copy number variation in a genome from a small population or individual. The method provides for pre-amplification of the gene of interest in the sample prior to analysis by digital PCR. The pre-amplification step distributes the distribution of individual gene copies to individual PCR reaction samples so that they are detected in a manner that represents the actual copy number rather than determined by digital PCR without pre-amplification. sell.

  The digital PCR-based method of the present invention has the ability to discriminate less than twice the difference in gene copy number with high accuracy. For example, it can distinguish 1, 2, 3 and 4 copies of a gene in different samples. In order to ensure that the apparent difference in gene copy number in different samples is real and is not distorted by the difference in sample amount, a term called relative copy number is used. The relative copy number of a gene (per human genome) can be expressed as the ratio of the copy number of the target gene to the copy number of the single copy reference gene in the DNA sample, which is usually 1. For example, the RNaseP gene is a single copy gene that encodes the RNA portion of the RNaseP enzyme and can be used as a reference gene in copy number assays.

  A commercially available digital array chip as shown in FIG. 3 for performing digital PCR is used to quantify DNA in the sample. The chip has 12 sample input ports for introduction of the sample mixture. Each sample mixture is divided into 765 reaction chambers in each of 12 panels. As described in the literature (see, for example, McBride et al., US Patent Application Publication No. 20050252773), the ability to quantify DNA in a sample is such that when an appropriate amount of DNA is introduced, a single DNA molecule Based on the fact that it is randomly distributed within the chamber.

  Quantify RNaseP and other genes in the same DNA sample simultaneously by using two assays for two genes (eg RNaseP and another gene of interest) with two fluorescent dyes on one chip A good estimate of the ratio of these two genes and the copy number of the gene of interest can be obtained.

  However, in the case of duplication, multiple copies of one gene are closely linked on the same chromosome and therefore may not be separated from each other even on the Digital Array. As a result, multiple copies behave as one molecule, and the total number of gene copies can be greatly underestimated.

  The present invention addresses this problem by including a pre-amplification step in the process. Pre-amplification is a PCR reaction with primers for both the gene of interest and a reference gene (eg RNaseP gene). This is typically done with a limited number of temperature cycles (eg, 10 cycles); assuming PCR efficiency is equal, the copy number of both genes is proportionally increased in the pre-amplification step. Using this process, even if multiple copies of a gene are linked on the genome, after pre-amplification, each copy of the gene of interest is amplified separately and divided into different chambers in the Digital Array. Since the newly generated molecules of both genes reflect the original ratio and are no longer linked, the digital chip analysis quantifies the molecules of the two genes and the ratio of the two genes (and hence the copy number of the target gene). ) Can be measured accurately.

Accordingly, the present invention provides a system and related methods for performing gene-based analysis. More specifically, the methods and systems of the invention generally relate to determining copy number variation of a polynucleotide of interest in a sample from a subject .

In one aspect, the invention is a method for determining the relative copy number of a target polynucleotide sequence in a subject 's genome;
a) pre-amplifying a target gene sequence and a reference gene sequence in a sample comprising genomic DNA of the subject ; thereby generating an amplified sample;
b) by distributing the product of (a) into a plurality of separate reaction volumes, amplifying the target and reference gene sequences in each reaction volume, and determining the relative amounts of the target and reference gene sequences in the amplified sample Performing a digital PCR, the method comprising the step of the relative amount of target and reference gene sequences in the amplified sample corresponding to the relative amount of target and reference gene sequences in the genome.

In a related aspect, the invention is a method for determining the relative copy number of a target polynucleotide sequence in a subject 's genome;
Pre-amplifying a target gene sequence and a reference gene sequence in a sample comprising genomic DNA of the subject ;
Assaying the target and reference gene sequences of the pre-amplified sample by digital PCR;
Determining (a) the number of amplified polynucleotide molecules comprising the target gene sequence and (b) the number of amplified polynucleotide molecules comprising the reference gene sequence and determining the ratio of (a) to (b) There is provided a method comprising the steps of:

In a related aspect, the invention is a method for determining the copy number of a target polynucleotide sequence in the genome of a subject ,
A step of performing a first polynucleotide amplification of a DNA sample obtained from a subject , wherein both a target polynucleotide sequence and a reference polynucleotide sequence having a predetermined genomic copy number N are amplified and thereby amplified. A prepared sample is generated; and
Distributing all or part of the amplified sample into a plurality of separate reaction volumes;
In each reaction volume, performing a second polynucleotide amplification, wherein the target polynucleotide sequence or a subsequence thereof is amplified if present, and the reference polynucleotide sequence or a subsequence thereof is amplified if present. Is a step;
Determining a reaction volume number A in which the target polynucleotide sequence or a subsequence thereof is present, and (b) determining a reaction volume number B in which the reference polynucleotide sequence or a subsequence thereof is present;
And the number of copies of the target polynucleotide in the genome is approximately equal to (A) / (B) × N.

In some embodiments, the sample is from a human. In certain embodiments, the ratio is about 0.5 of (a) to (b), the ratio of one of the on chromosome has deletion of (a), or (a) to (b) is about 1 a .5, there is an overlap of on one of the chromosomes (a). In some embodiments, the ratio of the target gene sequence to the reference gene sequence that deviates substantially from a value of 1 indicates an abnormal target gene sequence copy number in the patient's genome.

  In some embodiments, performing the first polynucleotide amplification comprises combining the biological sample with a composition comprising a primer specific for the target polynucleotide sequence and a primer specific for the reference polynucleotide sequence. And conducting a polymerase chain reaction (PCR) assay to separately amplify the target and reference polynucleotides in substantially equal proportions.

  In some embodiments, the first polynucleotide amplification has between 4 and 15 temperature cycles. In some embodiments, the reaction volume is disposed in a microfluidic device and the first polynucleotide amplification is performed in a reaction volume separated from the microfluidic device.

  In some embodiments, prior to the dispensing step, all or part of the amplified sample is combined with reagents selected for amplification of the target gene sequence and the reference gene sequence. Usually a portion is used and the amplified sample can be diluted before dispensing a portion into the reaction volume. In some embodiments, the amplification is PCR amplification.

In some embodiments, the reference gene sequence amplification primer used in the first polynucleotide amplification step is the same as that used in the second polynucleotide amplification step . In some embodiments, the target gene sequence amplification primer used in the first polynucleotide amplification step is the same as that used in the second polynucleotide amplification step . In some embodiments, the reagent comprises a first probe that selectively hybridizes to a target gene sequence, and a second probe that selectively hybridizes to a reference gene sequence under conditions suitable for polynucleotide amplification. Including. In some embodiments, the first and second probes comprise different detectable labels, and binding of the first or second probe during polymerase chain reaction (PCR) based polymerization, or the first or second Probe degradation results in a change in the detectable fluorescence of each detectable label.

In some embodiments, the reference gene sequence comprises a polynucleotide sequence that at least partially encodes an RNaseP enzyme, beta-actin or GAPDH. In some embodiments, determining the relative copy number of the target gene sequence includes detecting loss of heterozygosity in the subject 's genome. In some embodiments, a ratio of target gene sequence to reference gene sequence having a value substantially greater than or less than 1 indicates loss of heterozygosity in the patient's genome.

For a more complete understanding of the nature and advantages of the present invention, reference should be made to the following detailed description taken together with the accompanying figures. The drawings illustrate embodiments of the invention. The present invention can be modified in various ways without departing from the present invention. Accordingly, the drawings / drawings and descriptions of these embodiments are exemplary in nature and not limiting.
Accordingly, the present invention provides the following items:
(Item 1)
A method for determining the relative copy number of a target polynucleotide sequence in a subject's genome, the method comprising:
Preamplifying a target gene sequence and a reference gene sequence in a sample comprising genomic DNA of the subject;
Assaying the target gene sequence and the reference gene sequence of the pre-amplified sample by digital PCR;
Determining (a) the number of amplified polynucleotide molecules comprising the target gene sequence, and (b) the number of amplified polynucleotide molecules comprising the reference gene sequence, the ratio of (a) to (b) Steps to determine and
Including a method.
(Item 2)
Item 2. The method according to Item 1, wherein the sample is from a human.
(Item 3)
2. The method of item 1, wherein the ratio of (a) to (b) is about 0.5 and there is a deletion of (a) on one chromosome.
(Item 4)
Item 2. The method according to Item 1, wherein the ratio of (a) to (b) is about 1.5, and there is an overlap of (a) on one chromosome.
(Item 5)
A method for determining the copy number of a target polynucleotide sequence in the genome of a subject,
Performing a first polynucleotide amplification of a DNA sample obtained from a subject, wherein both the target polynucleotide sequence and the reference polynucleotide sequence having a predetermined genomic copy number N are amplified, thereby Generating an amplified sample; and
Distributing all or part of the amplified sample into a plurality of separated reaction volumes;
Performing a second polynucleotide amplification in each reaction volume, wherein the target polynucleotide sequence or a subsequence thereof is amplified, if present, and the reference polynucleotide sequence or a subsequence thereof is present If amplified, the step;
Determining a reaction volume number A in which the target polynucleotide sequence or a subsequence thereof is present, and (b) determining a reaction volume number B in which the reference polynucleotide sequence or a subsequence thereof is present;
Wherein the copy number of the target polynucleotide in the genome is approximately equal to (A) / (B) × N.
(Item 6)
Performing the first polynucleotide amplification comprises combining the biological sample with a composition comprising a primer specific for the target polynucleotide sequence and a primer specific for a reference polynucleotide sequence; 3. The method of item 2, comprising performing a polymerase chain reaction (PCR) assay to separately amplify the reference polynucleotide in substantially equal proportions.
(Item 7)
Item 7. The method according to Item 6, wherein the first polynucleotide amplification comprises 4 to 15 cycles.
(Item 8)
The method of item 2, wherein the reaction volume is disposed in a microfluidic device and the first polynucleotide amplification is performed in a reaction volume separate from the microfluidic device.
(Item 9)
Item 3. The method according to item 2, wherein all or part of the amplified sample is combined with a reagent selected for quantitative amplification of the target gene sequence and the reference gene sequence before the distributing step.
(Item 10)
10. The method of item 9, wherein the reference gene sequence amplification primer used in the first polynucleotide amplification step is the same as that used in the second polynucleotide amplification step.
(Item 11)
Item 11. The method according to Item 10, wherein the target gene sequence amplification primer used in the first polynucleotide amplification step is the same as that used in the second polynucleotide amplification step.
(Item 12)
10. The item 9, wherein the reagent comprises a first probe that selectively hybridizes to a target gene sequence under conditions suitable for polynucleotide amplification and a second probe that selectively hybridizes to a reference gene sequence. the method of.
(Item 13)
The first and second probes comprise different detectable labels and are suitable for binding of the first or second probe or degradation of the first or second probe during polymerase chain reaction (PCR) based polymerization. 13. The method of item 12, wherein the result is a change in detectable fluorescence of each of the detectable labels.
(Item 14)
2. The method of item 1, wherein the reference gene sequence comprises a polynucleotide sequence that at least partially encodes an RNaseP enzyme, beta-actin or GAPDH.
(Item 15)
3. The method of item 1 or 2, wherein the ratio of target gene sequence to reference gene sequence that deviates substantially from a value of 1 indicates an abnormal target gene sequence copy number in the genome of the patient.
(Item 16)
3. The method of item 1 or 2, wherein determining the relative copy number of the target gene sequence includes detecting loss of heterozygosity in the genome of the subject.
(Item 17)
3. The method of item 1 or 2, wherein the ratio of target gene sequence to reference gene sequence having a value substantially greater than or less than 1 indicates loss of heterozygosity in the patient's genome.

FIG. 1 is a flow diagram illustrating the general steps of the method of the invention described herein. FIG. 2A shows an exemplary channel design of a microfluidic device, according to an embodiment of the invention. FIG. 2B shows an exemplary channel design of a microfluidic device, according to an embodiment of the invention. FIG. 3 is a simplified diagram of a microfluidic device according to an embodiment of the invention. FIG. 4A shows a portion of the microfluidic device shown, for example, in FIG. FIG. 4B shows a portion of the microfluidic device shown, for example, in FIG. FIG. 4C shows a portion of the microfluidic device shown, for example, in FIG. FIG. 6 shows exemplary copy number variation results performed using a microfluidic device. FIG. 5 shows the results of an exemplary loss of heterozygosity performed using a microfluidic device. 4 is a graph illustrating detection of loss of heterozygosity according to one embodiment of the invention. FIG. 3 is a schematic diagram showing a partial result of a fictitious experiment in which the copy number of a target sequence T is determined. Shown is a reaction volume of 64 × 64 matrix in which the target sequence was amplified and detected using the VIC labeled (yellow) probe and the single copy reference sequence was amplified and detected using the FAM labeled (green) probe. Nineteen reaction volumes with a yellow label and twelve reaction volumes with a green label were detected, indicating a ratio of approximately 1.5 (19/12 = 1.58≈1.5), and the diploid genome This shows that there are three copies of the target sequence.

  The method and system of the present invention for determining the copy number of a target polynucleotide sequence in a patient's genome, including copy number variation associated with a genetic disease. In particular, the methods and systems described herein can be used to detect target polynucleotide copy number variation in a patient's genome using genomic material present in a patient-derived sample. The techniques of the present invention typically use a polynucleotide amplification-based assay to determine the relative copy number of a target polynucleotide sequence and a reference polynucleotide sequence in a sample. Genomic copy number is known per reference sequence. Thus, the copy number of the target polynucleotide can be analyzed relative to the reference polynucleotide to determine the relative copy number of the target polynucleotide. Target and / or reference polynucleotide sequences are sometimes referred to as “genes”. However, it will be appreciated that the term “gene” does not necessarily indicate that the sequence encodes a protein (or RNA).

  Copy number detection and analysis techniques are consistently suitable for so-called “digital analysis” or “digital PCR”, such as microfluidic devices that contain a large number or density of small volume reaction sites (eg, nano-volume reaction sites or reaction volumes). Throughput devices can be used. Accordingly, the copy number variation detection and analysis techniques of the present invention can be employed between hundreds to thousands of reaction volumes disposed in a reaction / assay platform or microfluidic device that includes the exemplary devices described herein. And dispensing or dividing the sample.

The methods of the invention include a pre-amplification step in which DNA from a biological sample (eg, genomic DNA) is amplified using polymerase chain reaction (PCR) or other quantitative amplification techniques. Typical biological samples include cells (including lysed cells and cell homogenates homogenates), serum, and biological fluids. Although the methods herein are generally described with respect to human DNA (eg, to determine copy number variation in the genome of a human patient), it will be appreciated that the method has variations in the amount of genetic material. It can be modified / applied for any sample. For example, the method can be used for genetic analysis of animals, plants, bacteria and fungi, and genetic analysis of human subjects. Methods for collecting and processing biological samples containing DNA are known and need not be discussed here. In the assays of the present invention, DNA can be isolated from cells or biological fluids, or the assay can be performed using, for example, cell lysates containing DNA. Thus, as used herein, a “DNA sample” can mean a purified, semi-purified, unpurified form of DNA, particularly genomic DNA. As used herein, “obtaining a DNA sample from a subject ” simply refers to the fact that the DNA sample is the starting material for a subsequent analysis step (eg, a pre-amplification step). “Obtaining a DNA sample” does not mean, for example, the act of collecting cells from a subject or isolating DNA, but may simply obtain a tube containing pre-collected DNA.

  FIG. 1 shows the general steps for carrying out the method described herein. In one exemplary embodiment, the steps of the method include pre-amplification comprising an assay primer, a suitable buffer system, nucleotides, and a DNA polymerase enzyme (eg, a polymerase enzyme modified for “hot start” conditions). Providing a master mix; adding genomic DNA to the pre-amplification master mix; pre-amplifying the target sequence (s) and reference sequence; and digital PCR analysis of the pre-amplified sequence (endpoint assay). Or in a real-time assay) and comparing the frequency of the target sequence (s) against the frequency of the reference sequence. It will be appreciated that FIG. 1 is provided to assist in understanding the present invention, but is not intended to limit the present invention.

In pre-amplification, which is the first step in FIG. 1, a first polynucleotide amplification of a DNA sample obtained from a subject is performed. In the pre-amplification step, both the target polynucleotide sequence and the reference polynucleotide sequence are amplified. Methods for PCR amplification are well known and need not be described here.

  In some embodiments, the target sequence is a sequence whose deletion or duplication is related to the phenotype of interest. In some embodiments, the target sequence is a sequence whose deletion or duplication is not related to a known phenotype of interest, but for which information about the distribution or correlation of variation in a particular population is sought.

  A reference sequence is a sequence having a known (or putative) genome copy number. Thus, the reference sequence is not likely to be amplified or deleted in the genome. It is not necessary to determine the reference sequence copy number experimentally in each assay. Rather, the copy number can be estimated based on the normal copy number in the organism of interest. For example, one useful reference sequence in the human genome is the sequence of the RNase P gene, which is a single copy gene present in two copies per diploid genome (having one copy number per haploid genome). Other useful reference sequences for purposes of illustration include β-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), but it should be understood that the invention is not limited to a particular reference sequence. .

Pre-amplification can be performed as a PCR reaction using primers for both the RNaseP (reference gene) and the target gene of interest. The reaction is typically carried out in a limited number of temperature cycles (eg, 5 cycles, or 10 cycles). In some embodiments, the optimal number of cycles depends on the PCR efficiency of the reference gene and the target gene. In certain embodiments, the number of temperature cycles during the pre-amplification assay can range from about 4-15 temperature cycles, or from about 4-10 temperature cycles. In some embodiments, the number of temperature cycles can be greater than 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 15.

  The pre-amplification reaction is preferably quantitative or proportional. That is, the relative number (ratio) of target and reference sequence amplicons should reflect the relative number (ratio) of target and reference sequences in the genomic (or other) DNA being amplified. Methods for quantitative amplification are known techniques. For example, Arya et al., 2005, Basic principals of real-time quantitative PCR, Expert Rev Mol Diag. 5 (2): 209-19. In the case of duplicate genes, the primer must be selected so that each duplicate copy of the target gene of interest is amplified separately. Thus, after selective pre-amplification and sample distribution into separate reaction volumes, a proportional number of amplicons corresponding to each sequence is distributed into the reaction volume. Since the newly generated molecules of both genes reflect the original ratio, the number of molecules of the target gene and the reference gene can be quantified by subsequent copy number analysis. As a result, the ratio of the two genes can be accurately measured. Since the copy number of the reference sequence is known, the copy number of the target sequence can be determined.

It is desirable that the amplification efficiencies of the target and reference sequences are similar or nearly equal in order not to introduce any bias in the ratio of the two gene copies. Therefore, primer pairs and amplification conditions must be selected to obtain such results. The amplification efficiency of any primer pair can be easily determined using conventional techniques (see, eg, Furtado et al., “Application of real-time quantitative PCR in the generation of gene expression. (See Wymondham, Norfolk, UK: Horizon Bioscience p. 131-145 (2004))
While it is desirable that the amplification efficiencies of the target and reference sequences be approximately equal, any efficiency can be achieved by a limited number of preamplification temperature cycles (typically less than 15, usually less than 10 or 10, most often about 5). By greatly reducing the difference, the effect of the normal difference on the result becomes insignificant.

As described above, the amplification method is a known technique. For illustration, the reaction mixture used in the pre-amplification method (pre-amplification composition or mix) is a suitable buffer, a magnesium ion (Mg 2+) source in the range of about 1 to about 10 mM, preferably in the range of about 2 to about 8 mM. , Nucleotides, and optionally surfactants and stabilizers. An example of one suitable buffer is TRIS buffer at a concentration of about 5 mM to about 85 mM, with a concentration of 10 mM to 30 mM being preferred. In one embodiment, the TRIS buffer concentration is in the form of 20 mM , double strength (2X) in the reaction mix. The reaction mix has a pH range of about 7.5 to about 9.0, with a pH range of about 8.0 to about 8.5 being typical. The concentration of nucleotides ranges from about 25 mM to about 1000 mM, and can typically range from about 100 mM to about 800 mM. Examples of dNTP concentrations are 100, 200, 300, 400, 500, 600, 700, and 800 mM. Surfactants such as Tween ™ 20, Triton ™ X100, and Nonidet ™ P40 can also be included in the reaction mixture. Stabilizers such as dithiothreitol (DTT, Cleland reagent) or mercaptoethanol may also be included. The pre-amplification reaction mix includes primers for the pre-amplification reaction. The primer is generally the same sequence that is used in the PCR assay after the sample is prepared, but the concentration is generally low. The primer concentration can be higher, equal or lower than the primer concentration used in the PCR assay. Embodiment, about 50,25,20,10 or 5 times greater than the primer concentration of the PCR assay higher, equal to, or one-tenth, one twentieth of 1 35 minutes, 1/50, 65 min 1, 1 75 minutes of the 1, 100-minute, 1 125 min, 1 150 min, 1 175 min, and the use of 200 minutes 1 a primer is of. Primers used in pre-amplification can include random primers, poly A tails, and specific primers designed for the PCR assay of interest.

  The reaction mix can optionally include a reference dye to normalize the results of subsequent real quantitative PCR analysis. An example of a common commercially available reference dye is ROX. A commercially available reaction mix containing ROX dye is available from CellsDirect 2X Reaction Mix, Cat. Nos. 11754-100 and 11754-500, available from Invitrogen Corporation.

A DNA polymerase enzyme (eg, Taq polymerase) is also added to the reaction mix. In one embodiment, Taq polymerase, such as Platinum® Taq DNA, is a recombinant Taq DNA polymerase complexed with an antibody that inhibits polymerase activity at ambient temperature. After a denaturation step in PCR, complete polymerase activity is restored, providing a “hot start”.

Pre-amplified samples prepared by the methods of the present invention are particularly suitable for digital PCR analysis and for distinguishing chromosomal duplications of genes. In particular, pre-amplified samples are assayed in multiple low volume PCR experiments. In digital PCR, the same (or substantially similar) assay is performed on a sample of genomic DNA. The number of individual reactions for a given genomic sample can vary from about 2 to over 1,000,000. Preferably, the number of assays performed on the sample is 100 or more, more preferably 200 or more, more preferably 300 or more. Larger scale digital PCR where the number of assays performed on the sample is 500 or more, 700 or more, 765 or more, 1,000 or more, 2500 or more 5,000 or more 7,500 or more, or 10,000 or more Can also be done. The number of assays performed can be up to about 25,000, up to about 50,000, up to about 75,000, up to about 100,000, up to about 250,000, up to about 500,000, up to about 750 per genomic sample. , 1,000, up to about 1,000,000, or more than 1,000,000 assays, etc. The amount of DNA used in digital PCR is generally selected such that there is one nucleic acid fragment or less in each digital PCR reaction.

As shown in FIG. 1, following a pre-amplification step, a sample (or portion thereof) containing a pre-amplification product with proportionally amplified genetic material (eg, amplicons corresponding to target and reference polynucleotide sequences) Are distributed into separate locations or reaction volumes, such that each reaction well contains, for example, an average of about 1 or less amplicons per volume. Thus, most reaction volumes do not have amplicons but have one target sequence amplicon or one reference sequence amplicon. Dilute (typically 1:10 to 1:20) the pre-amplified sample to adjust the concentration of amplicon to remain at zero or one amplicon per reaction volume (on average) and / or Or it is generally useful to use a small portion of the amplified sample. In some cases, the product of the pre-amplification step can be used without adding additional amplification reagents (eg, polymerase), but it is generally useful to add new reagents for amplification, optionally including different primers. . Thus, the biological sample can be combined with reagents selected for quantitative or non-quantitative amplification of both the target polynucleotide sequence and the reference polynucleotide 12 before or after dispensing (step 2).

  In addition, the pre-amplification step is generally a PCR-type amplification, but the second amplification (ie, amplification of the amplicon sequence generated by the pre-amplification) is not limited to, for example, but a Nasba (Compton, 1991. Nucleic Acid Sequence). -Amplification of Nature 350: 91-91, 1991) and the Eberwine protocol (Van Gelder et al., Amplified RNA synthesized from limited heterogeneous S.Ac. It can be broken.

  As noted above, it will be appreciated that the amount of DNA template and amplicon (a function of starting genomic DNA amount, number of amplification cycles, amplification efficiency and reaction volume size) is adjusted to achieve the desired partition. One skilled in the art can determine the concentration of amplicon in the pre-amplification product and calculate the appropriate amount for the input. More conveniently, a series of serial dilutions of the pre-amplification product can be tested. For example, the device shown in FIG. 3 (commercially available from Fluidigm Corp. as BioMark 12.765 Digital Array) allows 12 dilutions to be tested simultaneously. Optionally, the optimal dilution can be determined by generating a linear regression plot. For optimal dilution, the line should be straight and pass through the origin. The original sample concentration can then be calculated from the plot.

  After dispensing, genomic material contained in multiple reaction chambers can be amplified to further perform a sample assay to determine the number of reaction volumes in which amplicons corresponding to target or reference sequences have been sequestered (FIG. 1). 14). The second amplification can be performed using the same primer used in the pre-amplification, or a different primer (eg, a nested set).

In order to distinguish the signal originating from the target polynucleotide relative to the reference polynucleotide, differential detection and analysis of the sample can be performed (FIGS. 1, 16). For example, a separate reaction site analysis can be used to calculate the ratio of the reaction volume containing the target polynucleotide sequence to the reaction volume containing the reference polynucleotide sequence. The methods include genetic detection of target sequences in the subject 's genome, including detection of gene deletions or duplications, loss of heterozygosity, etc., eg, aneuploidy (eg, trisomy) and many other genetic abnormalities. The method may further include detecting and analyzing relevant information. Further details regarding method steps, including various fractional detection and analysis techniques, are provided below.

As disclosed above, samples containing pre-amplified products or unamplified genetic material can be distributed to separate locations or reaction volumes of the detection and analysis platform. Sample distribution may be performed using a variety of techniques and devices, such as, for example, flow-based distribution in a microfluidic device including multiple small volume reaction sites / chambers. In general, the dispensing steps of the methods described herein are performed to separate the sample material of interest, eg, target and reference sequences, into individual reaction sites for later detection and analysis.

  Within each of the plurality of reaction sites or volumes, one or more amplification assays can be performed, including multiplex reaction detection quantitative analysis / amplification of the target polynucleotide sequence and the selected reference polynucleotide sequence. The ratio of detection sequences in a sample can be calculated using detection techniques such as digital PCR analysis, real-time PCR curve monitoring and / or comparison of endpoint images of positive reaction chambers of one assay versus another assay. Alternatively, the concentration (copy number / μL) of any sequence in the DNA sample is calculated using the number of positive reaction chambers in the device containing at least one copy of that sequence, and to calculate the copy number, The ratio of the target and reference sequence concentrations can be determined. See co-pending US patent application Ser. No. 12 / 170,414, “Method and Apparatus for Determining Copy Number Variation Digital PCR,” which is incorporated herein by reference for all purposes. Dube et al., 2008, “Mathematical Analysis of Copy Number Variation in a DNA Sample Using Digital PCR on a Nanofluidic E2”, which is incorporated herein by reference for all purposes. doi: 10.1371 / journal. pone. See also 0002876.

  As noted above, the present invention includes methods and amplification-based techniques for determining target polynucleotide copy number variation, for example, in a patient's genome, and in some cases for subsequent quantitative amplification and analysis. A pre-amplification step may be performed prior to sample distribution in the microfluidic device. Pre-amplification is desirable, for example, when multiple copies of a single target gene are in close proximity on the same chromosome, so that the target sequence cannot be optimally divided during quantitative analysis, for example when distributed in a microfluidic device. Can be done. In such cases, multiple copies of the target gene can be undercounted or quantified as a single molecule rather than two. Therefore, the total number of gene copies can be underestimated.

In accordance with the present invention, CNV calculations are performed in order to conveniently distinguish the apparent difference in gene copy numbers in different samples from distortions such as distortions caused by differences in sample volume or assay noise / errors. It typically includes a “number” calculation. The relative gene copy number (per genome) can be expressed as the ratio of the copy number of the target gene to the copy number of the single copy reference gene in a DNA sample of known concentration (copy number) in the patient's genome. This is typically equal to 1. By utilizing two assays for two genes (target and reference polynucleotides) with two different labels (eg, fluorescent dyes) on the same digital array, the methods described herein can be performed. In use, both genes in the same DNA sample can be quantified simultaneously. Alternatively, although less convenient, the target amplicon (from pre-amplification) is amplified on one reaction volume set of chips, and the test amplicon (from pre-amplification) is assayed in a different amplicon set. The data can be compared. The ratio of these two genes is the target polynucleotide sequence in the DNA sample, or the relative copy number of the gene of interest. In one approach, the method, the target polynucleotide sequence or steps and, reference polynucleotide sequence or reaction volume number subsequence thereof is present to determine the reaction volume number (A) of the partial sequence is present (B ) and steps of determining, when N is genomic copy number of a predetermined reference sequence, as a step of determining that the number of copies of the target polynucleotide is approximately equal to (a) / (B) × N in the genome Can be summarized. Of course, while most polyploidy is low in most organisms (eg, humans usually have two copies of somatic chromosomes), the number of amplicons detected in the present invention inherently introduces experimental error. Therefore, (A) / (B) × N is almost related to the copy number. For example, (A) can be experimentally determined to be 936, (B) can be experimentally determined to be 596, and N can be 1 per haploid genome. (A) / (B) × N is equal to 1.57 (about 1.5), which is understood to indicate that there are about 1.5 copies of A per haploid genome ( Ie trisomy of A). See FIG. 8 and the examples below.

  Various detection platforms or microfluidic devices and methods can be used in the practice of the present invention. In some embodiments, the device can be constructed using a variety of materials such as glass, plastic, silicon, elastic polymers (eg, polydimethylsiloxane, polyurethane, or other polymers). In certain embodiments of the present invention, the microfluidic devices used to practice aspects of the present invention are typically constructed at least partially from an elastomeric material, and include monolayer and multilayer soft lithography (MSL) technology and / or Or constructed by sacrificial layer encapsulation methods (eg, Unger et al., 2000, Science 288: 113-116, and WO 01/116, all incorporated herein by reference for all purposes). 01025). Using such a method, a microfluidic device is designed in which solution flow through the device flow channel is at least partially controlled by one or more control channels separated from the flow channel by an elastomeric membrane or segment. Can be done. This membrane or segment can be deflected or retracted into the flow channel with the control channel by exerting an actuation force on the control channel. By controlling the degree to which the membrane is deflected into or drawn from the flow channel, the solution flow through the flow channel can be slowed or completely blocked. Using this type of control and flow channel combination, various types of valves to regulate solution flow, as detailed in Unger et al., Supra, WO 02/43615 and 01/01025. And you can prepare the pump.

  Sample distribution in the microfluidic devices described herein can be performed in part by certain properties of elastomeric materials generally recognized in the prior art. For example, Allcock et al. (Contemporary Polymer Chemistry, 2nd Ed.) Generally describe “elastomers” or “elastomeric materials” as polymers that exist at temperatures between their glass transition temperature and melting temperature. Elastomeric materials allow the polymer chains to easily twist, allowing the main chain to uncoil in response to force and to re-coil the main chain in the absence of force to take its original form. , Exhibit elasticity. In general, elastomers deform when a force is applied, but return to their original shape when the force is removed. The elasticity exhibited by the elastomeric material is characterized by Young's modulus. Elastomeric materials utilized in the microfluidic devices disclosed herein are typically between 1 Pa-1 TPa, in other cases between about 10 Pa-100 GPa, and in other cases about 20 Pa-1 GPa. And in other cases between about 50 Pa-10 MPa and in some cases between about 100 Pa-1 MPa. Elastomeric materials having Young's modulus outside these ranges can also be utilized depending on the needs of the specific case.

  Given the considerable diversity of polymer chemistry, precursors, synthetic methods, reaction conditions, and additive candidates, a variety of properties can be selected for applications and applications. Thus, in the context of the present invention, there are a number of possible elastomer systems that can be used to make monolithic elastomeric microvalves and pumps. Some of the microfluidic devices described herein are made from elastic polymers such as GE RTV 615 (formulation), vinyl silane cross-link (type) silicone elastomer (family). However, the microfluidic system of the invention is not limited to this one formulation, type or even family of polymers, but rather almost any elastic polymer is suitable. The choice of material typically depends on the specific material properties (eg, solvent resistance, hardness, gas permeability, and / or temperature stability) required for the application being made. For further details regarding the types of elastomeric materials that can be used in the manufacture of the elements of the microfluidic devices disclosed herein, see Unger et al. (2000) Science 288: 113-116, and WO 02/43615 and 01/01025. Which are incorporated herein by reference in their entirety for all purposes.

  Device manufacturing and temperature cycling.

  As indicated, the techniques of the present invention may incorporate the use of a variety of detection platforms, including high throughput microfluidic devices suitable for digital analysis or digital PCR. Device fabrication, system element aspects, and temperature cycling aspects are further detailed below.

In one embodiment, microfluidic devices suitable for use with the present invention can be constructed utilizing single layer and multilayer soft lithography (MSL) techniques and / or sacrificial layer encapsulation methods. One basic MSL approach involves casting a series of elastomeric layers into a micromachined mold, removing the layers from the mold, and fusing the layers. In the sacrificial layer encapsulation approach, a pattern of photoresist is deposited where the channel is desired. These techniques and their use in making microfluidic devices are described, for example, by Unger et al. (2000) Science 288: 113-116, and Chou et al., Each incorporated herein by reference in its entirety for all purposes. (2000) "Integrated Elastomer Fluidic Lab-on-a-chip-Surface Pattering and DNA Diagnostics," in Proceedings of the Solid State Actor and Henstor and Sensor. C. And WO 01/01025.

Briefly, the exemplary manufacturing method described above involves first photolithography using a photoresist (Shipley SJR5740) to form an upper layer (eg, an elastomer layer with a control channel) and a lower layer (eg, an elastomer with a flow channel). A step of manufacturing a matrix for the layer) on a silicon wafer. The channel height can be accurately controlled by the spin coating speed. A photoresist channel is formed by developing the photoresist after exposure to ultraviolet light. Thermal reflow process and protection treatment is incorporated by reference herein in its entirety, M. A. Unger, H .; -P. Chou, T .; Throsen, A.M. Scherer and S. R. This is typically accomplished as described by Quake, Science (2000) 288: 113. The mixed two-component silicone elastomer (GE RTV 615) is then spun into the bottom mold and poured onto the top mold. Spin coating can be utilized to control the thickness of the bottom polymer fluid layer. After baking in an oven at 80 ° C. for 25 minutes, the partially cured upper layer is peeled off from the mold and aligned with the lower layer. To bond these two layers irreversibly, a final baking at 80 ° C. for 1.5 hours is used. The RTV devices removed from the bottom silicon mother mold is, HCL in (0.1 N, 80 30 min at ° C.), are typically processed. This treatment acts to cleave a portion of Si-O-Si bonds, thereby exposed hydroxy groups increases the hydrophilic channels.

  The device can then optionally be sealed to the support. The support can be made of essentially any material, but since the seal formed is mainly due to adhesive forces, the surface must be flat to ensure a good seal. Examples of suitable support materials include glass, plastic and the like.

As a result of the device being formed by the method described above, the carrier (eg glass slide) forms one wall of the flow channel. Alternatively, the device once removed from the matrix is sealed with a thin elastomeric membrane so that the flow channel is completely encapsulated in the elastomeric material. The resulting elastomeric device can then be optionally matched to a carrier support.

Layer Formation In one embodiment, microfluidic devices, including those in which reagents are deposited at the reaction site during manufacture, are formed from three layers. The lower layer is the layer on which the reagent is deposited. The underlayer can be formed from a variety of elastomeric materials as described in the references cited above for the MLS process. Typically, the material is a polydimethylsiloxane (PDMS) elastomer. Based on the location and position of the reaction site desired for a particular device, the position on the lower layer where the appropriate reagent should be spotted can be determined. Since PDMS is hydrophobic, the deposited aqueous spot shrinks to form a very small spot. As described above, since the reagent introduced at the reaction site is intended to dissolve in the sample solution, the arbitrarily deposited reagent is deposited such that no covalent bond is formed between the reagent and the elastomeric surface. The

  The other two layers of the device are the layer where the flow channel is formed, and the layer where the control channel and optionally the guard channel are formed. These two layers are prepared according to the general method described earlier in this section. The resulting bilayer structure is then placed on the first layer on which the reagent is deposited. Specific examples of the composition of the three layers are as follows (assignment of component A to component B): first layer (sample layer) 30: 1 (weight); second layer (flow channel layer) 30: 1; And third layer (control layer) 4: 1. However, it is anticipated that other compositions and ratios of elastomer components may be utilized as well. During this process, the reaction site is aligned with the deposited reagent so that the reagent is placed within the appropriate reaction site.

  According to the present invention, temperature cycling can be performed on a microfluidic device. In particular, temperature cycling can be used to perform an amplification reaction that facilitates analysis of the sample dispensed in the reaction chamber.

  A variety of different sophistication options are available for temperature control within selected regions of the microfluidic device or across the device. Thus, as used herein, the term temperature controller refers to one or more microfluidic devices or a portion of a microfluidic device (eg, within a particular temperature range or in a matrix of blind channel type microfluidic devices. It broadly means a device or element that can adjust the temperature (at the junction).

  In general, to temperature cycle the device, the device is placed on a temperature cycling plate. For example, ThermoHybaid Px2 (Franklin, MA), MJ Research PTC-200 (South San Francisco, Calif.), Eppendorf Part # E5331 (Westbury, NY), Techne Part # 205330 (Prince), Are readily available from commercial sources.

  In order to ensure the accuracy of the temperature cycling step, in some devices it is useful to incorporate sensors that sense temperature in various regions of the device. One structure for detecting temperature is a thermocouple. Such thermocouples can be made as thin film wires patterned into the underlying substrate material or as wires incorporated directly into the microfabricated elastomer material itself.

  Various means of temperature detection / monitoring can be included in the system / device of the present invention. For example, the temperature may be detected by a change in electrical resistance. Thermochromic materials are another type of structure that can be used to detect the temperature over the area of an amplification device. Another approach to detecting temperature is by using an infrared camera. Yet another approach to temperature detection is by using pyroelectric sensors. Other electrical phenomena, such as capacitance and inductance, can be utilized to detect temperature according to embodiments of the present invention. Using the known equations for thermal diffusivity and the appropriate values of elastomer and glass utilized in the device, the time required for the temperature in the reaction site to reach the temperature that the controller is trying to maintain can be calculated.

In addition to various material compositions and properties that may be suitable, microfluidic devices suitable for use with the present invention may include various features, designs, channel structures, and the like. The device typically includes a plurality of “flow channels” that generally mean a flow path through which the solution can flow. Further, the device may include a “control channel” or a channel designed to couple with the flow channel so that it can be used to operate the flow within the flow channel. The device can further include features for further regulating fluid flow, such as a “valve”, in which the flow channel and control channel intersect and deflect into the flow channel in response to an actuation force It is, or are separated by retracted may elastomeric membrane can include configuration. Certain embodiments may also include “vias” that refer to channels formed in the elastomeric device to provide fluid access between the external port of the device and one or more flow channels. Thus, vias can serve as sample input or output, for example.

Many types of channel structures or designs can be implemented in the present invention. As shown in FIG. 2A, one type of channel design that can be included in the device of the present invention includes an open channel design. “Open channel” or “open end channel” refers to a flow channel disposed between separate vias such that the flow channel has an outlet (eg, outlet) and a separate inlet (eg, inlet). In general, open-channel network design is connected around one or more branches flow channels may form an open channel network, including a flow channel via or inlet countercurrent least two pairs. One or more valves formed by adjacent / overlapping control channels may be actuated to separate separate regions of the branch channel and form reaction sites. Such a valve provides a mechanism for switchably separating a plurality of reaction sites. As described herein, a device may include one or more open flow channels from which one or more channels diverge. One or more reaction regions or reaction sites can be located anywhere along the length of the flow channel. A valve formed by the overlapping flow channels may be actuated to separate the reaction site (s) located along the channel, thereby providing a mechanism for switchably separating the reaction sites . Thus, each device can contain a large number of reaction sites (eg, 10,000+) and can achieve a high reaction site density, thereby significantly increasing the size of these devices compared to conventional microfluidic devices. Reduction is possible. An open channel design may have, for example, a branch flow channel that can be addressed from more than one location / via. This design aspect allows fluids to enter from different directions, for example if a particular channel / branch flow channel is obstructed or blocked (eg due to manufacturing variations, defects, etc.) This can be particularly advantageous because the channel can be filled to the side. In contrast, channels that can only be accessed from one end with an occlusion can only fill up to the point of obstruction or blockage, and if the reaction site exists beyond the occlusion, those sites become unusable. sell.

  As shown in FIG. 2B, a microfluidic device suitable for use in accordance with the present invention may utilize a “blind channel” or “blind fill” design. Such devices have one or more blind channels, i.e., a flow channel that has a termination or a single end so that solution can enter and exit the blind channel only from one end (i.e., there are no separate inlets and outlets for the blind channel). This can be partly characterized. These devices require only one valve for each blind channel to separate the blind channel regions to form isolated reaction sites. During the manufacture of this type of device, one or more reagents for performing the analysis are optionally deposited at the reaction site, which can result in a significant reduction in the number of inputs and outputs. Thus, a flow channel network in communication with the blind channel can be set up so that many reaction sites can be filled by a single or limited number of inlets (eg, less than 5 or less than 10). The ability to fill the blind flow channel is realized because the device is made from a sufficiently porous elastomeric material so that the air in the flow channel and blind channel can escape from these holes as the solution is introduced into the channel. The The lack of porosity of the materials utilized in other microfluidic devices eliminates the use of blind channel designs because there is no outlet for air in the blind channel when the solution is injected.

  In yet another embodiment, the microfluidic device of the present invention may optionally further include a guard channel in addition to the flow channel and valve or control channel. To reduce sample and reagent evaporation from the elastomeric microfluidic devices provided herein, multiple guard channels can be formed in the device. The guard channel is similar to the control channel in that it is typically formed in an elastomeric layer that overlaps the flow channel and / or reaction site. As such, the guard channel, like the control channel, is separated from the underlying flow channel and / or reaction site by a membrane or segment of elastomeric material. However, unlike the control channel, the guard channel has a considerably small cross-sectional area. In general, a membrane with a small area has less deflection than a membrane with a large area under the same pressure. The guard channel is designed to be pressurized to allow a solution (typically water) to flow into the guard channel. Water vapor originating from the guard channel can diffuse into the pores of the elastomer adjacent to the flow channel or reaction site, thus increasing the water vapor concentration adjacent to the flow channel or reaction site and reducing evaporation of the solution therefrom. For further discussion regarding guard channels located in microfluidic devices and suitable for use in accordance with the present invention, see McBride et al., US Patent Application Publication No. 20050252773, which is hereby incorporated by reference in its entirety for all purposes. See

  The device further includes a plurality of reaction sites or reaction volumes with which the reagents are reacted, and the device can incorporate various means (eg, pumps and valves) for selectively separating the reaction sites. The reaction site can be placed at any of a number of different locations within the device.

Because the device can include a relatively light transmissive elastomeric material, the reaction can be easily monitored using a variety of detection systems at essentially any location on the microfluidic device. When MSL-type devices are used, detection is most typically performed at the reaction site itself. The fact that such devices are manufactured from a substantially transparent material also means that certain detection systems can be utilized with current devices that cannot be used with conventional silicon-based microfluidic devices. Detector incorporated in the device, or separate from the device used in which aligned position with the detection region of the device, the detection can be achieved.

  In certain embodiments of the invention, the reaction volume is used with a mix or reagent that is first mixed (eg, mixed with a sample) and then introduced into the solution in a solution separated from the chip and other system elements. The reaction inside takes place.

  Devices are typically designed and set up to perform temperature controlled reactions such as temperature cycling amplification reactions. Thus, the device can be set / designed for temperature controlled reactions (eg, temperature cycling reactions) in the reaction volume. The device or part thereof, such as an elastomer device, can be secured to a support (eg, a glass slide). The resulting structure can then be placed on a temperature control plate, for example, to control the temperature at various reaction sites. In the case of a temperature cycling reaction, the device can be placed on any of a number of temperature cycling plates.

  As indicated above, any use of the microfluidic device to perform the method of the invention can be made using a variety of device characteristics and designs. The following description describes in more detail exemplary settings that can be used to perform a variety of analyzes, including analyzes that require temperature control (eg, nucleic acid amplification reactions). However, it should be understood that these settings are exemplary and modifications to these systems will be apparent to those skilled in the art.

  FIG. 3 is a simplified diagram of a microfluidic device, according to an illustrative embodiment of the invention. As shown in FIG. 3, a microfluidic device, also referred to as a digital array, can include a carrier 20 that can be made from materials that provide suitable mechanical support for various elements of the microfluidic device. For example, a device is made using an elastic polymer. The outer part of the device has the same footprint as a standard 384-well microplate, allowing independent valve operation. As described below, there are 12 input ports corresponding to 12 separate sample inputs to the device. The device has 12 panels 22 and each of the 12 panels can contain 765 6 nL reaction chambers with a total volume of 4.59 μL per panel. The microfluidic channel 24 can connect various reaction chambers on the panel to a fluid source, as described in detail below.

  The accumulator 26 can be pressurized to open and close the valve that connects the reaction chamber to the fluid source. As shown in FIG. 3, twelve inlets 28 may be provided for loading of the sample reagent mixture. In some applications, 48 inlets 28 are used to provide a source of reagents that are supplied to the biochip when the accumulator 26 is pressurized. In applications where no reagent is utilized, the inlet 28 and reagent side accumulator 26 may not be used. In addition, two inlets 30 are provided in the exemplary embodiment shown in FIG. 3 to provide hydration to the biochip. Hydration inlet 30 is in fluid communication with the device to facilitate control of the humidity associated with the reaction chamber. As will be appreciated by those skilled in the art, some elastomeric materials utilized in the manufacture of devices are gas permeable so that vaporized gas or vapor from the reaction chamber can pass from the elastomeric material into the ambient atmosphere. . In certain embodiments, fluid conduits installed in the peripheral portion of the device provide protection for hydrating liquids, such as buffers, or master mixes at the peripheral portion of the biochip that surrounds the reaction chamber panel, and thus reaction. Reduce or prevent evaporation of liquid present in the chamber. Thus, adding a volatile liquid, such as water, to the hydration inlet 30 can increase the humidity at the periphery of the device. In certain embodiments, a first inlet is in fluid communication with a hydration fluid conduit surrounding a panel on the first side of the biochip, and a second inlet is a hydration fluid tube surrounding the panel on the opposite side of the biochip. In fluid communication with the channel.

  While the above device and sample distribution is one exemplary system for performing the methods of the present invention, those of ordinary skill in the art will appreciate that many variations of the microfluidic device design described herein, Modifications and alternatives will be recognized. For example, the microfluidic device shown in FIG. 3 includes 12 panels each having 765 reaction chambers with a volume of 6 nL per reaction chamber, which is not required for the invention. The specific outline of the digital array depends on the specific application. Thus, for example, the scope of the present invention is not limited to a 12-panel digital array having 765 reaction chambers, and other combinations are within the scope of the present invention. Further description relating to digital arrays suitable for use in embodiments of the present invention is provided in US Patent Application Publication No. 2005/0252773, which is incorporated herein by reference.

  To run a large number of replicate samples, a significant amount of reagents can be required. In an embodiment of the present invention, digital PCR is performed on a small volume. The reaction chamber for performing low volume PCR can be from about 2 nL to about 500 nL. The smaller the reaction chamber volume, the greater the number of individual assays that can be performed (with different probes and primer sets, or as duplicates of the same probe and primer set, or arbitrarily swapped multiple replicates and many different assays. ). In one embodiment, the reaction chamber is about 2 nL to about 50 nL, preferably 2 nL to about 25 nL, more preferably about 4 nL to about 15 nL. In some embodiments, the reaction chamber volume is about 4 nL, about 5 nL, about 6, nL, about 7 nL, about 8, nL, about 9 nL, about 10 nL, about 11 nL, or about 12, nL. The sample chamber can be made of elastic polymers such as glass, plastic, silicon, polydimethylsiloxane, polyurethane, or other polymers. Samples processed by the method of the invention are suitable for copy number variation analysis using the BioMark ™ system (Fluidigm Corporation, South San Francisco, Calif.). The BioMark ™ system uses a polydimethylsiloxane microfluidic device that provides for the performance of multiple assays on multiple samples.

Fluidigm microfluidic devices (digital arrays) are manufactured by Fluidigm Corporation (South San Francisco, Calif.). Chips are manufactured by Multilayer Soft Lithography (MSL) (Unger MA, Chou HP, Thorsen T, Scherer A, Quake SR, Monolithic microfabricated valves and pumps. The chip has a sample channel with a semi-elliptical depth of an average of 10 μm, a width of 70 μm and a parallel spacing of 200 μm centered. Sample fluidics are manufactured using a two-layer molding process to create 265 μm (depth) × 150 μm × 150 μm split chambers placed along each sample channel. To another silicon over down layer, the control channel of the chip, running the sample channel and a right angle. The intersection of the channels forms a deflection valve for routing the fluid. During pressurization of the control channel, the thin film between layers blocks the sample channel and separates the individual division chambers. The control channel is 15 μm deep and 50 μm wide, with a parallel spacing of 300 μm between the hearts.

  Reaction mixes such as PCR mixes, sample mixes, pre-amplification product sample mixes are loaded into each panel and a single DNA molecule is randomly divided into various reaction chambers. After panel and reaction chamber loading, the digital array can be temperature cycled and then imaged with a suitable reader, such as a BioMark ™ instrument available from the assignee. The generated data is analyzed using Digital PCR Analysis software or other suitable analysis software available from the assignee. A further description of exemplary detection and / or analysis techniques suitable for use in embodiments of the present invention is provided in US Patent Application Publication No. 12 / 170,414, entitled “Copy Number Variation Determination by Digital PCR”; This is a co-pending application by the same applicant and is incorporated herein by reference for all purposes.

  4A-4C are simplified views of a portion of the device / biochip shown in FIG. FIG. 4A shows 12 panels 22, each of which includes a number of reaction chambers. FIG. 4B shows the outline of a number of reaction chambers 40 included in the panel. Reaction chambers 40 are spaced 200 μm apart as shown. FIG. 4C shows a fluorescent image of a portion of the panel. On the left side of the figure is the control section where all reaction chambers are shown dark. The right side of the figure shows a dark 42 appearance in a typical experiment where many of the reaction chambers did not produce significant fluorescence. However, a portion of the reaction chamber has fluorescent emission, indicating a “positive” reaction chamber 44. As described above in FIG. 2B, sample channels connect the individual reaction chambers and run from left to right, and control channels run from top to bottom in the lower layer. During pressurization of the control channel, the thin film between the layers blocks the sample channel and separates the individual reaction chambers. The valves divide individual chambers that remain closed during the PCR experiment.

  As described more fully throughout this specification, the chip is temperature cycled, imaged with a BioMark ™ real-time PCR system available from the assignee, and BioMark ™ available from the assignee. ) The number of positive chambers in each panel was counted using Digital PCR Analysis software such as Digital PCR Analysis. If two assays with two fluorescent dyes are used in a complex digital PCR reaction, the two genes can be quantified independently. In order to study copy number variation using a digital array, the ability to quantitate this gene independently is used as described herein. The number of genes that can be independently quantified in a PCR reaction depends on the number of fluorescent dyes and filters available.

As described in the general method steps above, after sample distribution, additional steps include an amplification step followed by detection and analysis of the results. In some embodiments of the invention, amplification and detection / analysis can be performed using a method that coordinates the two steps, such as quantitative PCR. In general, polynucleotides separated in each reaction site can be amplified, detected, and analyzed using a variety of possible strategies. One exemplary strategy involves amplifying the target and reference polynucleotides so that the amplified product can be used to determine the concentration of the target polynucleotide and the concentration of the reference polynucleotide. To perform amplification, reagents necessary for amplification are combined with the sample, and a first probe that selectively hybridizes to the target polynucleotide under conditions suitable for polynucleotide amplification, and selectively to the reference polynucleotide. A second probe that hybridizes may be included. The first and second probes can include different detectable labels to distinguish target and reference polynucleotide amplification products. Furthermore, the distinction between the target and reference polynucleotides can further provide a calculation of the concentration of the target nucleotide molecule as a ratio of the reference nucleotide molecules to determine the relative copy number of the target polynucleotide sequence in the subject 's genome. .

  The general amplification step prior to detection and analysis can be performed using a number of methods.

  In order to enhance understanding of the methods and systems described throughout the specification, technical terms are generally described below. The term “reagent” refers broadly to any agent used in a reaction. The reagent can include a single agent that can itself be monitored (eg, a substance that is monitored during heating), or a mixture of two or more agents. The reagent may be alive (eg, a cell) or not alive. Exemplary reagents for nucleic acid amplification reactions include, but are not limited to, buffers, metal ions, polymerases, primers, template nucleic acids, nucleotides, labels, dyes, nucleases and the like. Reagents for enzymatic reactions include, for example, substrates, cofactors, conjugated enzymes, buffers, metal ions, inhibitors and activators. Reagents for cell-based reactions include, but are not limited to, cells, cell-specific dyes, and ligands (eg, agonists and antagonists) that bind to cell receptors. Reagents can be included in the sample solution or can be optionally immobilized in a variety of ways (eg, covalently, non-covalently, via a suitable linker molecule). In an on-chip nucleic acid amplification reaction, for example, one or more reagents used in performing an extension reaction can be deposited at each reaction site during device fabrication (eg, by spotting).

  The term “label” refers to a molecule or embodiment of a molecule that can be detected by physical, chemical, electromagnetic, and other related analytical techniques. Examples of detectable labels that can be used include radioisotopes, fluorophores, chromophores, mass labels, high electron density particles, magnetic particles, spin labels, chemiluminescent emitting molecules, electrochemically active molecules, enzymes, Examples include, but are not limited to, cofactors, enzymes linked to nucleic acid probes, and enzyme substrates. The term “detectably labeled” refers to a unique property (eg, size, shape or color) that allows an agent to be conjugated to a label or to be detected without the agent being conjugated to a separate label. ).

The terms “nucleic acid”, “polynucleotide”, and “oligonucleotide” are used herein to include polymeric nucleotides of any length, including but not limited to ribonucleotides or deoxyribonucleotides. used. There is no intended length difference between these terms. Furthermore, these terms refer only to the primary structure of the molecule. Thus, in some embodiments, these terms, triplex can include double-stranded, and single stranded DNA, as well as triple-stranded, double-stranded, and single stranded RNA. These also include, for example, modified forms by methylation and / or capping, and unmodified forms of polynucleotides. More specifically, the terms “nucleic acid”, “polynucleotide”, and “oligonucleotide” include polydeoxyribonucleotides (including 2-deoxy-D-ribose), polyribonucleotides (including D-ribose), Any other type of polynucleotide that is an N- or C-glycoside of a purine or pyrimidine base, and other polymers containing non-nucleotide backbones such as polyamides (eg, peptide nucleic acids (PNA)) and polymorpholinos (Anti-Virals) , Inc., Corvallis, Oregon, commercially available as Neugene), and other synthetic sequence-specific nucleic acid polymers, such as those found in DNA and RNA, base pairing and base stacking Allow Provided that includes a configuration of a nucleobase that.

A “probe” is a nucleic acid that binds to a target nucleic acid of a complementary sequence by one or more types of chemical bonds, usually by complementary base pairing, usually by hydrogen bond formation, thus forming a duplex structure. is there. A probe binds or hybridizes to a “probe binding site”. Probe, particularly once the probe, its complementary target hybridizing Then, to enable the probe, the facile detection, can be labeled with a detectable label. The label attached to the probe can include any of a variety of labels known in the art that can be detected, for example, by chemical or physical means. Suitable labels that can be attached to the probe include radioisotopes, fluorophores, chromophores, mass labels, high electron density particles, magnetic particles, spin labels, chemiluminescent emitting molecules, electrochemically active molecules, enzymes, Cofactors and enzyme substrates are included but not limited to. Probes can vary greatly in size. Some probes are relatively short. Generally, a probe is at least 7-15 nucleotides in length. Other probes are at least 20, 30 or 40 nucleotides in length. Still other probes are slightly longer and are at least 50, 60, 70, 80, 90 nucleotides in length. Still other probes are longer, at least 100, 150, 200 or more nucleotides in length. The probe can be any particular length that falls within the aforementioned range.

A “primer” is in an appropriate buffer at an appropriate temperature and under an appropriate condition (ie, in the presence of an agent for polymerization, such as four different nucleoside triphosphates and a DNA or RNA polymerase or reverse transcriptase). ) template Ru Oh single-stranded polynucleotide can serve as a starting point for directed DNA synthesis. The appropriate length of the primer depends on the intended use of the primer, but is typically at least 7 nucleotides in length, more typically in the range of 10-30 nucleotides. Other primers are slightly longer, may be 30-50 nucleotides long, etc. Short primer molecules generally require lower temperatures to form sufficiently stable hybrid complexes with the template. The primer need not reflect the exact sequence of the template, but must be sufficiently complementary to hybridize with the template. The term “primer site” or “primer binding site” refers to the segment of target DNA to which a primer hybridizes. The term "primer pair", '3 end hybridizing'"downstream primer and 'a phase complementary strands hybridized with 5' end 5 of the amplified DNA sequence" upstream primer ", 3 sequences to be amplified "Means a primer set.

  A “fully complementary” primer has a completely complementary sequence over the entire length of the primer and has no mismatches. The primer is typically completely complementary to a portion (partial sequence) of the target sequence. “Mismatch” refers to a site where the nucleotides of the target nucleic acid aligned with the nucleotides of the primer are not complementary. The term “substantially complementary” when used in reference to a primer means that the primer is not completely complementary to its target sequence; instead, the primer is in each of its desired primer binding sites. It remains sufficiently complementary to selectively hybridize to the strand.

The term “complementary” means that one nucleic acid is identical to or selectively hybridizes to another nucleic acid molecule. When hybridization occurs is more selective than total lack of specificity, hybridization selectivity exists. Typically selected when there is at least about 55% identity, preferably at least 65%, more preferably at least 75%, and most preferably at least 90% identity in a stretch of at least 14-25 nucleotides Hybridization occurs. It is preferred that one nucleic acid specifically hybridizes to another nucleic acid. M.M. Kanehisa, Nucleic Acids Res. 12: 203 (1984).

  Detection occurs in the “detection section” or “detection region”. These terms and other related terms refer to the portion of the microfluidic device where detection occurs. As noted above, in devices that utilize certain designs (eg, open channel designs, blind channel designs, etc.), the detection section is typically a reaction site separated by a valve associated with each reaction site. The detection section of a matrix-based device is typically in the area of the flow channel adjacent to the intersection, the intersection itself, or the area that includes the intersection and the surrounding area.

  As described above, an exemplary copy number variation analysis can be performed using an on-chip quantitative PCR method. In particular, quantitative PCR may involve both polynucleotide amplification and amplification product detection / analysis. In addition to qPCR, various so-called “real-time amplification” or “real-time quantitative PCR” methods can also be used to determine the amount of target nucleic acid present in a sample by weighing the amount of amplification product formed during or after the amplification process itself. Can be used to determine the amount. The fluorogenic nuclease assay is one example of a real-time quantification method that can be successfully used with the devices described herein. This method of monitoring the formation of amplification products involves continuous measurement of PCR product accumulation using double-labeled fluorogenic oligonucleotide probes. This is an approach often cited in the literature as the “TaqMan” method.

  The probes used in such assays are typically short (eg, about 20-25 bases) polynucleotides labeled with two different fluorescent dyes. The 5 'end of the probe is typically attached to the reporter dye and the 3' end is attached to the quencher dye, although the dye may be attached at other positions on the probe. The probe is designed to have at least substantial sequence complementarity with the probe binding site on the target nucleic acid. Upstream and downstream PCR primers that bind to the region adjacent to the probe binding site are also included in the reaction mixture.

  When the probe is complete, energy transfer between the two fluorophores occurs and the quencher quenches the emission from the reporter. During the PCR extension step, the probe is cleaved by the 5 ′ nuclease activity of a nucleic acid polymerase such as Taq polymerase, which releases the reporter from the polynucleotide quencher and has a reporter emission intensity that can be measured with an appropriate detector. An increase occurs.

  One detector specifically configured for the measurement of fluorescence emission, such as that generated during a fluorogenic assay, is available from Applied Biosystems, Inc. of Foster City, CA. ABI 7700 manufactured by Computer software provided with the instrument can record the fluorescence intensity of the reporter and quencher during amplification. These recorded values can then be used to calculate continuously the normalized increase in reporter emission intensity and finally quantitate the amount of mRNA amplified.

  Additional details regarding the theory and practice of fluorogenic methods for making real-time measurements of the concentration of amplification products can be found in, for example, US Pat. No. 5,210,962, to Gelfund, each incorporated herein by reference in its entirety. No. 015, 5,538,848 to Livak et al., And 5,863,736 to Haaland, and Heid, C .; A. Et al., Genome Research, 6: 986-994 (1996); Gibson, U., et al. E. M, et al., Genome Research 6: 995-1001 (1996); Holland, P. et al. M.M. Et al., Proc. Natl. Acad. Sci. USA 88: 7276-7280, (1991); and Livak, K .; J. et al. Et al, described in PCR Methods and Applications 357-362 (1995). Therefore, as the amplification reaction proceeds, a larger amount of dye binds and is accompanied by an increase in the accompanying signal.

When performing on-chip amplification assays, during the temperature cycling process, for example, a plurality of primers each utilizing a specific target nucleic acid (eg, target polynucleotide sequence and reference polynucleotide sequence) are utilized. Thus, multiplex amplification can be performed within a single reaction site. The presence of different amplification products, by using a probe labeled differently for quantitative RT-PCR reactions, or can be detected using molecular beacons labeled differently (see supra). In such an approach, each differently labeled probe is designed to hybridize only to a particular amplified target. By judicious selection of the different labels utilized, an analysis can be performed in which different labels are excited and / or detected at different wavelengths in a single reaction. For further guidance relating to the selection of appropriate fluorescent labels in such an approach, see Fluorescence Spectroscopy (Edited by Pesce et al.) Marcel Dekker, New York, (1971); White et al. York, (1970); Berlman, Handbook of Fluorescence Spectra of Aromatic Molecules, 2 nd ed. , Academic Press, New York, (1971); Griffiths, Color and Constitution of Organic Modules, Academic Press, New York, (1976); Indicators (Bishhop). Pergamon Press, Oxford, 19723; and Haugland, Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Eugene (1992).

  When microfluidic devices such as open channel or blind channel design devices are utilized to perform nucleic acid amplification reactions, the reagents that can be deposited within the reaction site are necessary to perform the desired type of amplification reaction. It is a reagent. Usually this means that some or all of the following are deposited, eg, primers, polymerases, nucleotides, metal ions, buffers, and cofactors. In such a case, the sample introduced into the reaction site is a nucleic acid template. However, alternatively, a template may be deposited and amplification reagent poured into the reaction site. When a matrix device is utilized to perform the amplification reaction, the sample containing the nucleic acid template can be flowed into the vertical flow channel, the amplification reagent can be flowed into the horizontal flow channel, or vice versa.

In general, multiple genotyping and expression analysis can be performed at each reaction site, for example. A sample containing the target DNA can be introduced into a reaction site on the microfluidic device. In quantitative PCR methods such as TaqMan®, primers for amplifying different regions of the target DNA of interest are contained within a single reaction site. Product formed, in order to distinguish, for example, the target and reference polynucleotides, for each region, differently labeled probes are utilized. If an allele that is complementary to the probe is present in the target DNA, amplification occurs, which results in a detectable signal. Depending on which of the different signals is obtained, the nucleotide identity at the polymorphic site can be determined. If both signals are detected, both alleles are present. Temperature cycling during the reaction is carried out as described in the temperature control section above.

In some embodiments of the invention, differentially labeled probes complementary to each of the alleles can be included as reagents, along with primers, nucleotides and polymerases. However, the reaction can be performed with only one probe, but this can obscure whether the lack of signal is due to the absence of a particular allele or simply due to failure of the reaction. In the case of both typical alleles where two alleles are possible at the polymorphic site, two differently labeled probes, each completely complementary to one of the alleles, are usually amplified primers, nucleotides And included in the reagent mixture along with the polymerase.

  As shown by FIG. 4C, the signal from each reaction site can be detected and further analyzed to determine information about the sample. For example, samples processed by the methods of the invention can be obtained from the BioMark ™ system (Fluidigm Corporation, South San Francisco, Calif.). And suitable for use in copy number variation analysis using the BioMark ™ fluorescence imaging thermal cycler system. The BioMark ™ system uses a polydimethylsiloxane microfluidic device that provides for the performance of multiple assays on multiple samples.

  As described more fully throughout this specification, in some embodiments, the chip is temperature cycled and imaged with a BioMark ™ real-time PCR system available from the assignee, The number of positive chambers in each panel was counted using Digital PCR Analysis software such as BioMark ™ Digital PCR Analysis available from the assignee. If two assays with two fluorescent dyes are used in a complex digital PCR reaction, the two genes can be quantified independently. This ability to quantitate genes independently is used to study copy number variation using digital arrays, as described herein.

As generally described above, a reaction mix, such as a PCR mix, can be loaded into each panel and a single DNA molecule can be randomly divided into various reaction chambers. After panel and reaction chamber loading, the digital array is temperature cycled and then imaged with a suitable reader, such as a BioMark ™ instrument available from the assignee. The generated data is analyzed using Digital PCR Analysis software or other suitable analysis software available from the assignee.

  As described above, on-chip quantitative PCR can be used to perform certain embodiments of the invention. However, various detection strategies can be utilized with the microfluidic devices described above. The selection of an appropriate system is characterized in part by the type of device, the event to be detected and / or the type of drug. The detector is linked to a radioisotope, fluorophore, chromophore, high electron density particle, magnetic particle, spin label, chemiluminescent emitting molecule, electrochemically active molecule, enzyme, cofactor, nucleic acid probe It can be designed to detect a variety of signal types, including but not limited to signals from enzymes and enzyme substrates.

Exemplary detection methodologies include, but are not limited to, light scattering, multi-channel fluorescence detection, UV and visible wavelength absorption, emission, reflectance factor differences, and confocal laser scanning. Additional detection methods that can be used in certain applications include scintillation proximity assay techniques, radiochemical detection, fluorescence polarization, fluorescence correlation spectroscopy (FCS), time-resolved energy transfer (TRET), fluorescence resonance energy transfer (FRET). , And variations such as bioluminescence resonance energy transfer (BRET). Additional options for detection include electrical resistance, specific resistance, impedance, and voltage sensing .

  The detection section includes one or more microscopes, diodes, photostimulation devices (eg, lasers), photomultiplier tubes, processors and combinations of the foregoing that cooperate to detect signals associated with specific events and / or agents. I can contact you. In many cases, the detected signal is an optical signal that is detected in the detection section by an optical detector. The optical detector may include a fiber optic light guide that leads to one or more photodiodes (eg, avalanche photodiodes), eg, photomultiplier tubes, microscopes, and / or video cameras (eg, CCD cameras).

  The detector can be microfabricated in a microfluidic device or can be a separate element. If the detector is present as a separate element and the microfluidic device includes multiple detection sections, detection can occur within a single detection section at any time. Alternatively, a scanning system can be used. For example, certain automated systems scan the light source relative to the microfluidic device, and other systems scan the emitted light on the detector, or include a multi-channel detector. As a specific example, a microfluidic device can be mounted on a translatable stage and scanned under a microscope objective. The signal thus obtained is then sent to the processor for signal interpretation and processing. An array of photomultiplier tubes can also be utilized. In addition, an optical system can be utilized that has the ability to determine the signal from each section and collect signals from all the different detection sections simultaneously.

  Since the provided device is made entirely or mostly of a material that is light transmissive at the wavelength being monitored, an external detector can be used. This feature allows the devices described herein to utilize a number of optical detection systems that are not possible with conventional silicon-based microfluidic devices.

  In one embodiment, the detector uses a CCD camera and light path that provides a large field of view and high numerical aperture to maximize the amount of light collected from each reaction chamber. In this regard, the CCD is not used to generate an image of the array, but is used as an array of photodetectors, each pixel or group of pixels corresponding to a reaction chamber. Thus, the optics can be modified so that the image quality is reduced or defocused to increase the depth of field of the optical system and more light is collected from each reaction chamber.

The detector can include a light source for stimulating a reporter that produces a detectable signal. The type of light source utilized will depend in part on the nature of the reporter being activated. Suitable light sources include, but are not limited to, lasers, laser diodes, and high intensity lamps. If a laser is utilized, the laser can be utilized to scan an entire set of detection sections or a single detection section. Laser diodes can be microfabricated in the microfluidic device itself. Alternatively, the laser diode can be fabricated in another device that is placed adjacent to the microfluidic device utilized to perform the temperature cycling reaction so that the laser light from the diode is directed to the detection section.

  Detection can also involve a number of non-optical approaches. For example, the detector may also include, for example, a temperature sensor, a conductivity sensor, a potentiometric sensor (eg, a pH electrode) and / or an amperometric sensor (eg, monitoring oxidation and reduction reactions).

  A number of commercially available external detectors can be utilized. Many of these are fluorescence detectors because it is easy to prepare fluorescently labeled reagents. Specific examples of available detectors include, but are not limited to, Applied Precision Array WoRx (Applied Precision, Issquah, WA).

  In some embodiments, a FRET based detection method is used. This type of detection method involves detecting a change in fluorescence from a donor (reporter) and / or acceptor (quencher) fluorophore in a donor / acceptor fluorophore pair. The donor and acceptor fluorophore pair is selected so that the emission spectrum of the donor overlaps the excitation spectrum of the acceptor. Thus, energy transfer from the donor to the acceptor can occur when the fluorophore pair is brought sufficiently close to each other. This energy transfer can be detected. See U.S. Patent No. 5,945,283 and WO 97/22719.

Molecular beacons provide a particularly useful approach. Due to molecular beacons, a change in the conformation of the probe when hybridized to the complementary region of the amplification product results in the formation of a detectable signal. The probe itself contains two sections: one section at the 5 ′ end and the other section at the 3 ′ end. These sections are adjacent to and complementary to the section of the probe that anneals to the probe binding site. One end section is typically attached to a reporter dye and the other end section is usually attached to a quencher dye.

  In solution, the two end sections can hybridize to each other to form a hairpin loop. In this conformation, since the reporter and quencher dye are sufficiently close together, the fluorescence from the reporter dye is effectively quenched by the quencher dye. In contrast, a hybridized probe produces a linearized conformation that reduces the degree of quenching. Therefore, it is possible to indirectly monitor the formation of amplification products by monitoring the luminescence changes for the two dyes. This type of probe and its use are described, for example, in Piatek, A .; S. Nat. Biotechnol. 16: 359-63 (1998); Tyagi, S .; And Kramer, F .; R. , Nature Biotechnology 14: 303-308 (1996); and Tyagi, S .; Nat. Biotechnol. 16: 49-53 (1998), each incorporated herein by reference in its entirety for all purposes.

  Other well known amplification / detection methods (for illustration and not limitation) include Invader (Neri, BP, et al., Advances in Nucleic Acid and Protein Analysis 3826: 117-125, 2000) Nasba (see, eg, Compton, J. Nucleic Acid Sequence-based Amplification, Nature 350: 91-91, 1991); Scorpion (Thelwell N. et al., Nucleic Acids Research 1, 2000; 37:52; 76:52); Capacitive DNA Detection (see, for example, Sonn et al., 2000, Proc. Natl. Acad. Sci. U. S.A. 97: 10687-10690). Each of these references is incorporated herein by reference for all purposes.

  As noted above, the methods of the invention include performing various reaction / amplification assays that require various reagents, compositions, buffers, additives, and the like. The reaction mixture can be prepared at least in part, separately from the assay platform or microfluidic chip / device, or in the reaction site of the device itself (eg, spotting). Certain reaction mixtures or compositions can be prepared and included as part of a kit or system. For example, the system can include a pre-amplification mixture / composition, an amplification assay composition, and a microfluidic device for performing amplification and copy number detection assays. Two or more elements of the system can be combined and provided as part of a kit or system.

Reactions performed by the microfluidic devices disclosed herein include various reagents, buffers, compositions, additives, etc. that can be prepared to perform the reactions of the present invention (eg, pre-amplification, quantitative amplification, etc.). Can be used. Thus, for example, in the case of a device on which reagents are deposited, the reagents can be spotted with one or more reactants at the reaction site, for example. In other embodiments, reagents may be provided in a mix or reagent volume independent of the chip or other system elements, for example when no on-chip spotting is performed. One set of additives is a blocking reagent that blocks protein binding sites on the elastomeric substrate. A variety of such compounds can be utilized, including a number of different proteins (eg, albumin proteins such as gelatin and various bovine serum albumins) and glycerol. Surfactant additives may also be useful. Any of a number of different surfactants can be utilized. Examples include, but are not limited to, SDS and various Triton surfactants.

In particular cases of nucleic acid amplification reactions, a number of different types of reagents and / or additives may be included. One category is enhancers that promote amplification reactions. Such additives, drugs (e.g., betaine) reducing the secondary structure in nucleic acids, and mistakes priming event to reduce drug (such as tetramethylammonium chloride) is included but not limited thereto.

  In general, CNV calculations can be based on "relative copy number" so that the apparent difference in gene copy number in different samples is not distorted by the difference in sample amount. The relative copy number (per genome) of a gene can be expressed as the ratio of the copy number of the target gene to the copy number of the single copy reference gene in the DNA sample, which is typically 1. By using two assays for two genes (target polynucleotide sequence and reference polynucleotide sequence) with two different fluorescent dyes on the same device, both genes in the same DNA sample can be quantified simultaneously. . Therefore, the ratio of the two genes is the relative copy number of the target nucleotide sequence in the DNA sample.

  In one embodiment of the invention, pre-amplification can be performed using a reference gene such as RNaseP, which is a single copy gene encoding the RNA portion of the RNaseP enzyme, ribonucleoprotein.

  Running a large number of replicate samples can require a significant amount of reagents. In an embodiment of the present invention, digital PCR is performed on a small volume. The reaction chamber for performing low volume PCR can be from about 2 nL to about 500 nL. The lower the reaction chamber volume, the greater the number of individual assays that can be performed (with different probes and primer sets, or as duplicates of the same probe and primer set, or arbitrarily swapped multiple replicates and many different assays. ). In one embodiment, the reaction chamber is about 2 nL to about 50 nL, preferably 2 nL to about 25 nL, more preferably about 4 nL to about 15 nL. In some embodiments, the reaction chamber volume is about 4 nL, about 5 nL, about 6, nL, about 7 nL, about 8, nL, about 9 nL, about 10 nL, about 11 nL, or about 12, nL. The sample chamber can be constructed from elastic polymers such as glass, plastic, silicon, polydimethylsiloxane, polyurethane, or other polymers. Samples processed by the method of the invention are suitable for copy number variation analysis using the BioMark ™ system (Fluidigm Corporation, South San Francisco, Calif.). The BioMark system uses a polydimethylsiloxane microfluidic device that provides for the execution of multiple assays on multiple samples.

The Fluidigm device / nanofluidic chip (digital array) and BioMark fluorescence imaging temperature cycler system are manufactured by Fluidigm Corporation (South San Francisco, Calif.). The exemplary chip shown in FIG. 5 has 12 panels, each of which includes 765 6 nL chambers with a total volume of 4.59 μL per panel. The chip is manufactured according to the Multilayer Soft Lithography (MSL) methodology. Unger MA, Chou HP, Thorsen T, Scherer A, Quake SR. Monolithic microfabricated valves and pumps by multilayer soft lithography. Science. 2000; 288: 113-116. The chip has a sample channel with an average semi-elliptical depth of 10 μm, a width of 70 μm, and parallel spacing of 200 μm. Sample fluidics are manufactured using a two-layer molding process to create 265 μm (depth) × 150 μm × 150 μm split chambers placed along each sample channel. In another silicon chromatography emission layers, the control channel of the chip, running the sample channel and a right angle. The intersection of the channels forms a deflection valve for determining the course of the fluid. During pressurization of the control channel, the thin film between layers blocks the sample channel and separates the individual division chambers. The control channel is 15 μm deep and 50 μm wide, with a parallel spacing of 300 μm between the hearts. The outer part has the same footprint as a standard 384-well microplate, allowing independent valve operation. There are 12 input ports, corresponding to 12 separate sample inputs to the chip. The chips used can incorporate 765 6nL split chambers per sample input for a total of up to 14,400 chambers per chip. In this particular embodiment, sample channels connect the individual reaction chambers from left to right and control channels are from top to bottom in the lower layer. During pressurization of the control channel, the thin film between the layers blocks the sample channel and separates the individual reaction chambers. The valves divide individual chambers that remain closed during the PCR experiment.

To perform a real-time PCR reaction, a master amplification mix (eg, a “master mix”) is combined with a sample containing the product of the pre-amplification assay. The master mix includes a suitable buffer, a magnesium ion (Mg2 +) source in the range of about 1 to about 10 mM, preferably in the range of about 2 to about 8 mM, nucleotides, and optionally surfactants and stabilizers. An example of a suitable buffer is a TRIS buffer at a concentration of about 5 mM to about 85 mM, with a concentration of 10 mM to 30 mM being preferred. In one embodiment, the TRIS buffer concentration is 20 mM in double strength (2X) form in the reaction mix. The reaction mix can have a pH range of about 7.5 to about 9.0, with a pH range of about 8.0 to about 8.5 being typical. The concentration of nucleotides can range from about 25 mM to about 1000 mM, and typically ranges from about 100 mM to about 800 mM. Examples of dNTP concentrations are 100, 200, 300, 400, 500, 600, 700, and 800 mM. Surfactants such as Tween ™ 20, Triton ™ X100, and Nonidet ™ P40 can also be included in the reaction mixture. Stabilizers such as dithiothreitol (DTT, Cleland reagent) or mercaptoethanol may also be included.

DO WE NEED THIS PARAGRAPH? Furthermore, the master mix can optionally include dUTP as well as uracil DNA glycosylase (uracil-N-glycosylase, UNG). UNO is the product of the Escherichia coli ung gene. cloned, sequenced and expressed in E. coli. Uracil-DNA-N-glycosylase (UNG) from DNA (single- and double-stranded), DNA sugar - uracil residues were removed without destroying the phospho backbone, thus, as a hybridization target, Or prevent its use as a template for DNA polymerase. The resulting abasic site is susceptible to hydrolytic cleavage at high temperatures. Thus, removal of uracil bases is usually accompanied by DNA fragmentation. Duncan, B.M. K. And Chambers, J .; A. (1984) GENE 28, 211, Varshney, U .; , Hutcheon, T .; And van de Sande, J. et al. H. (1988) 1. Biol. Chem. 263,7776. Master mixes are commercially available from Applied Biosystems, Foster City, CA (TaqMan (R) Universal Master Mix, cat. Nos. 4304437, 4318157, and 4326708). The use of UNG is typically limited to digital PCR assays and not used in pre-amplification assays.

  In complex applications, different fluorescent reporter dyes are used to label separate primers or probes for quantification of different genes. In relative expression studies using complex PCR, the amount of primer of the reference gene (eg, β-actin or GAPDH) should be limited to avoid competition between reference and sample gene amplification. In general, the final concentration of the reference gene primer should be between 25 and 100 nM. Primer titration can be useful for optimization.

  In one exemplary embodiment of the invention, the copy number of CYP2D6 was determined with or without pre-amplification. Using pre-amplification, it was found that CYP2D6 in one sample had an overlap (copy number 3), but the same sample showed a copy number 2 when it was not accompanied by pre-amplification.

A PCR master mix useful for performing PCR assays on samples prepared by the methods of the present invention can be prepared with the following composition: 20 mM Tris, pH 8.0, 100 mM KCl, 1% Glycerol, 0.04% Tween. _{(TM), 5mM MgCl 2, 400mM dNTP} , 0.08U / μL AmpliTaq ( TM) Gold enzyme (Applied Biosystems, Foster City, CA ). AmpliTaq DNA polymerase is a recombinant form of Taq DNA polymerase. This is because E.I. It is obtained by expressing the Taq DNA polymerase gene in an E. coli host. It lacks endonuclease and 3′-5 ′ exonuclease activity, but has 5′-3 ′ exonuclease activity, like natural Taq DNA polymerase.

  Pre-amplification in one example is GeneAmp PCR system 9700 (Applied Biosystems, CA), IxPreAmp master mix (Applied Biosystems, CA), 225 nM primer (RNaseP as reference polynucleotide) and target sequence of interest, and 1 μL of DNA. Performed in a 5 μL reaction containing the sample. Temperature cycling conditions were 95 ° C., 10 minutes hot start and 10 cycles 95 ° C. for 15 seconds, 60 ° C. for 1 minute. 20 μL of water was added to each reaction after pre-amplification and samples were analyzed on a digital array.

Five Coriell DNA samples were analyzed on a digital chip. The number of CYP2D6 and RNaseP molecules in the same volume (4.59 μL) of each sample was counted using the BioMark Digital PCR Analysis software using Poisson correction and the Simant algorithm (see above, Dube et al.). A representative heat map is shown in FIG. 5 in a simplified black and white view. For purposes of illustration, shown in white, black, and gray , events are recorded and yellow, green corresponding to RNaseP gene (VIC, yellow), CYP2D6 gene (FAM, red), and no gene, respectively. Or visually as a color such as red. A no template control (NTC) was performed on panels 1 and 12.

  A ratio of the number of CYP2D6 gene molecules to the RNaseP gene was obtained for five samples. Two of the ratios are about 0.5, which means that there is only one copy of the CYP2D6 gene in each cell of these two samples (RNaseP is a single copy gene, each There are always two copies of the gene in the cell). Thus, the individual from which the DNA sample was collected should have a deletion of the CYP2D6 gene on one chromosome. The other three samples had a ratio of about 1, but this does not exclude the possibility of duplication as the two closely linked replicas are on one molecule and cannot be separated. A pre-amplification reaction was performed on these five samples and the pre-amplification products were analyzed on a digital chip (Table 2).

^* Sample NA11994 has a CYP2D6 gene duplication on one chromosome.

  As shown in Table 2, when genomic DNA is used, two samples with a ratio of CYP2D6 to RNaseP of about 0.5 are still about 0 when the pre-amplification process of the present invention is used. A ratio of .5 was given. A ratio of 0.5 indicates a deletion. Two samples with a ratio of about 1 when genomic DNA was used also had a ratio of about 1 with the pre-amplification product, indicating a normal allelic state. However, one sample that had a ratio of about 1 when genomic DNA was analyzed had a ratio of 1.5 when the pre-amplification process was used. This indicates that the sample has a CYP2D6 gene duplication.

Detection of loss of heterozygosity One useful application of the described method for determining copy number variation of a particular gene of interest includes detection of loss of heterozygosity (LOH). The techniques disclosed herein can provide a new level of sensitivity and flexibility in detecting reduced heterozygosity. Exemplary applications include detection and / or abnormal X chromosome copy number, or aneuploidy studies. Loss of heterozygosity (LOH) refers to a change from a heterozygous state in the normal genome to a homozygous state in the paired tumor genome. Investigations show that loss of the entire X chromosome is associated with a number of cancers. Moertel, CA. Et al., Cancer Genet. Cytogenet. 67: 21-27 (1993). For example, 40% of ovarian cancers involve LOH in the X chromosome region. Osbourne, R.A. J. et al. And Leech, V .; , Br. J. et al. Cancer 69: 429-438 (1994). Also, an increase in the X chromosome has been shown to be relatively common in leukemias and lymphomas. Sandberg AA. "The X chromosome in human neoplasia, including sex chromatin and congenital conditions with X-chromosome anomalies.In:Sandberg AA, editor.Cytogenetics of the mammalian X chromosome, part B: X chromosome anomalies and their clinical manifestations.New York: Alan R Liss, 459-98 (1983).

To perform LOH experiments, the microfluidic devices described herein can be provided. FIG. 3 shows the structure of an exemplary device that was used in one example to determine the loss of heterozygosity (see, eg, the above description for further details of the device). Briefly, the device includes an integrated fluid circuit (IFC) having 12 panels, each with a flow input for a sample or assay mixture. In one embodiment, the sample is moved to the tip for loading the digital array placed on IFC controller, using a software interface is pressure loaded assay components into separate panels of 765 reactions Was loaded by. Each of the twelve samples premixed with the master mix and primer-probe set was dispensed into separate inlets on the chip frame. In each panel, one sample was divided into 765 individual 6 nL real-time PCR reactions. PCR was performed on the samples. A digital array was placed on a real-time PCR system for temperature cycling and fluorescence detection. Results from the experiment were viewed and analyzed using BioMark® application software. To compare one assay with another, a real-time PCR curve or a positive chamber end point image was recorded, for example, the ratio of any two sequences in a DNA sample was calculated. Digital arrays offer improved linearity, sensitivity, and ease of use for analysis.

  In the described examples, DNA from cell lines containing 1, 2, 3, 4 or 5 copies of the X chromosome was obtained (Coriell Institute for Medical Research, Camden, NJ). Using a digital array, each sample is a single copy target, three separate X chromosome TaqMan® primer-probe sets-FAM label 123B, SMS, co-amplified in the presence of the VIC label “reference” sequence. And YY2 (BioSearch Technologies, Novato, CA)-.

FIG. 6 shows a black and white view showing color-based results to check for loss of heterozygosity as described, further illustrating each test performed in duplicate panels in a digital array. FIG. 6 also shows a close-up view of the panel 4 of the device. In each panel, the number of target positive (light gray, corresponding to one color, eg yellow) and reference positive (dark gray, second color, eg red) chambers are counted. Corrected for multiple dyes per chamber. From these results, the raw ratio of target to reference was determined. A no template control (NTC) was used in panels 1 and 12. Of course, in practice it is possible to record the results that the experiment showed in different colors and colors, such as red and yellow, but this is shown as gray in the black and white figure in FIG.

A simple linear fitting was used to determine the copy number. FIG. 7 shows the average of three separate assay ratios (Y axis) plotted against the known X chromosome copy number (X axis), including error bars indicating the standard error of the mean. The ratio produced a slope of the DNA sample known to contain 1, 2, 3, 4 or 5 copies of the X chromosome. The individual raw ratio measurements were multiplied by 2 to give an average copy number per diploid genome. The average response for all assays with 1-5 copy number variants was an r ² value of 0.994, indicating high linear assay performance.

Table 3 lists the raw ratios from the TaqMan® primer probe set for individual X chromosome tests performed on the microfluidic device. By multiplying the average ratio by 2, the average X chromosome copy per genome was determined. The last column on the right shows the standard error of the mean (SEM). As shown in Table 3, the average copy per genome corresponded well with the known X chromosome copy number of the sample.

These results show that the methods and devices described herein allow for the detection and differentiation of small but biologically relevant gene copy number differences in highly complex genomic DNA samples. Samples selected for these tests are described in Visakorpi et al., 1994, Am. J. et al. Pathol, 145: 624-630 and Pinkel et al., 1998, Nat. Genet. 20: 207-211, similar or identical to those tested in CGH assays and MIP-based microarray studies. The results of using the method of the present invention with a digital array reduce manual technical operations, thus requiring less effort, increasing efficiency, and copying at least as differentially as known CGH and MIP methods. A number estimate can be generated. Moreover, the ability to perform multiple TaqMan® assays in a digital PCR format provides both biological robustness and assay redundancy and compensates for inter-assay amplification differences. If multiple loci are targeted simultaneously, the overall assay results are valid even if there is a single mutation or deletion in the local primer-probe binding site. In addition, efficacy can be enhanced by using a pre-amplification step before transferring the sample onto the microfluidic device for analysis.

While the invention has been described with reference to the above examples, it will be appreciated that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the full scope of the following claims and their equivalents.

Claims (19)

A method for determining the relative copy number of a target polynucleotide sequence in the genomic DNA of an organism, the method comprising:
Pre-amplifying a target polynucleotide sequence and a reference polynucleotide sequence in a sample comprising genomic DNA of the organism;
Assaying the target polynucleotide sequence and the reference polynucleotide sequence of the pre-amplified sample by digital PCR;
Determining (a) the number of amplified polynucleotide molecules comprising the target polynucleotide sequence and (b) the number of amplified polynucleotide molecules comprising the reference polynucleotide sequence; (a) vs. (b) Determining the ratio of.   The method of claim 1, wherein the organism is a human.   The method of claim 1, wherein the organism is a non-human animal.   The method of claim 1, wherein the organism is a plant.   2. The method of claim 1, wherein the ratio of (a) to (b) is 0.5 and there is a deletion of the target polynucleotide sequence on one chromosome.   2. The method of claim 1, wherein the ratio of (a) to (b) is 1.5 and there is an overlap of the target polynucleotide sequence on one chromosome. A method for determining the copy number of a target polynucleotide sequence in the genomic DNA of an organism,
Performing a first polynucleotide amplification of a DNA sample obtained from the organism, wherein both the target polynucleotide sequence and the reference polynucleotide sequence, wherein the reference sequence has a predetermined genomic copy number N, are amplified thereby Generating an amplified sample; and
Distributing all or part of the amplified sample into at least 5000 separated reaction volumes;
Performing a second polynucleotide amplification in each reaction volume, wherein the target polynucleotide sequence or a subsequence thereof is amplified, if present, and the reference polynucleotide sequence or a subsequence thereof is present If amplified, the step;
Determining a reaction volume number A in which the target polynucleotide sequence or a subsequence thereof is present, and determining a reaction volume number B in which the reference polynucleotide sequence or a subsequence thereof is present;
Wherein the copy number of the target polynucleotide in the genome is approximately equal to (A) / (B) × N.   Performing the pre-amplification comprises combining the sample with a composition comprising a primer specific for the target polynucleotide sequence and a primer specific for the reference polynucleotide sequence; and a target polynucleotide and a reference polynucleotide 7. A method according to any one of claims 1 to 6, comprising the step of performing polymerase chain reaction (PCR) to separately amplify at substantially equal rates. 8. The method of claim 7 , wherein the reaction volume is disposed in a microfluidic device and the pre-amplification is performed in a reaction volume separate from the microfluidic device.   Prior to the step of assaying the target polynucleotide sequence and the reference polynucleotide sequence of the pre-amplified sample by digital PCR, all or part of the pre-amplified sample is converted to a target polynucleotide sequence and a reference. 7. A method according to any one of claims 1-6, combined with a reagent selected for quantitative amplification of the polynucleotide sequence.   9. A primer specific for the reference polynucleotide sequence used in the pre-amplification step is also used in the step of assaying the reference polynucleotide sequence of the pre-amplified sample by digital PCR. The method described in 1.   12. A primer specific for the target polynucleotide sequence used in the pre-amplification step is also used in the step of assaying the target polynucleotide sequence of the pre-amplified sample by digital PCR. The method described in 1.   The reagent comprises a first probe that selectively hybridizes to a target polynucleotide sequence and a second probe that selectively hybridizes to a reference polynucleotide sequence under conditions suitable for polynucleotide amplification. 10. The method according to 10.   The first probe and the second probe comprise different detectable labels, the binding of the first probe or the second probe during polymerase chain reaction (PCR) based polymerization, or the first probe or the 14. The method of claim 13, wherein degradation of the second probe results in a change in detectable fluorescence of the respective detectable label.   2. The method of claim 1, wherein the reference polynucleotide sequence comprises a polynucleotide sequence that at least partially encodes an RNaseP enzyme, beta-actin or GAPDH.   The ratio of target polynucleotide sequence to reference polynucleotide sequence that substantially deviates from a value of 1 indicates an abnormal target polynucleotide sequence copy number in the genome of the subject. The method according to item. The reference polynucleotide sequence has a predetermined genomic copy number N;
Determining the number of amplified polynucleotide molecules comprising the target polynucleotide sequence comprises determining the number A of reaction volumes in which the target polynucleotide sequence or a subsequence thereof is present;
Determining the number of amplified polynucleotide molecules comprising the reference polynucleotide sequence comprises determining the number B of reaction volumes in which the reference polynucleotide sequence or a subsequence thereof is present;
The method of claim 1, wherein the relative copy number of the target polynucleotide in the genome is approximately equal to (A) / (B) × N.   8. The method of claim 7, comprising distributing all or part of the amplified sample into at least 10,000 separate reaction volumes. The method of claim 7, wherein the reaction volume is 2 nL to 50 nL.